Stiffness-engineered hydrogels drive neural regeneration via the Man2b1– lysosomal mechanotransduction axis

Peripheral nerve repair requires biomaterials capable of dynamically guiding neural cells. While stiffness-tunable hydrogels hold promise, how their mechanical properties translated into pro-regenerative intracellular signals remains poorly understood. To address gap, we developed a library of polyacrylamide/chitosan hydrogels with tunable stiffness (1.5∼39 kPa) to mimic the native neural microenvironment. Using this platform, we identified an optimal stiffness (∼13.75 kPa) that maximizes PC12 cell adhesion, spreading, neurite outgrowth, and migration. When fabricated into nerve guidance conduits and implanted in rat sciatic nerve defects, hydrogels with this optimal stiffness promoted axonal regeneration and functional recovery to a significantly greater extent than their softer or stiffer counterparts. Transcriptomic analysis further revealed coordinated upregulation of lysosomal and focal adhesion-related genes, with the lysosomal α-mannosidase Man2b1 identified as a core mechanosensitive regulator. We demonstrated that Man2b1 converts optimal matrix stiffness into enhanced lysosomal activity and stable nanoscale adhesion complexes (e.g., FAK, Vinculin). Importantly, genetic ablation of Man2b1 disrupted this mechano-transduction cascade and abolished the pro-regenerative effects of the optimized hydrogel both in vitro and in vivo. Collectively, this study establishes stiffness-engineered hydrogels as a robust platform for peripheral nerve repair and uncovers a fundamental mechano-transduction axis-the Man2b1-lysosome-adhesion signaling cascade-that governs neural regeneration. Our findings highlight how rational biomaterial design can precisely modulate cellular machinery to advance functional tissue engineering. STATEMENT OF SIGNIFICANCE: Peripheral nerve injuries often lead to permanent disability due to the limited regenerative capacity of adult neurons and the lack of biomaterials that can effectively guide repair. While substrate stiffness is known to influence cell behavior, translating this knowledge into effective nerve guides requires both identifying an optimal mechanical range and understanding the underlying cellular mechanisms. This study addresses this dual challenge by first engineering a tunable chitosan-based hydrogel platform to discover a pro-regenerative stiffness. More importantly, we leverage this material system to uncover a previously unknown mechano-transduction pathway-the Man2b1-lysosome-adhesion axis-that is essential for translating optimal mechanical cues into neuronal growth and regeneration. This work therefore provides not only a promising material strategy for advanced nerve guides but also a fundamental mechanobiological principle that could inform the design of biomaterials for a wide range of regenerative applications.